Nordic BioSite AB Propellervägen 4A, 183 62 Täby, Sverige T +46 (0)8 544 433 40, F +46 (0)8 756 94 90 [email protected], www.nordicbiosite.com Org. Nr: 556539-9374, Säte: Täby

Anti-/CCNA2 Antibody

Catalog Number ABB-2326 Lot number see vial Size 100μg/vial

Description Clonality Polyclonal Form Lyophilized Formulation Each vial contains 5mg BSA, 0.9mg NaCl, 0.2mg Na2HPO4, 0.05mg NaN3. Specificity No cross-reactivity with other . Species Reactivity Human, Mouse, Rat Immunogen E.coli-derived human Cyclin A2 recombinant (Position: A10-K168). Human Cyclin A2 shares 74.5% amino acid (aa) sequence identity with mouse Cyclin A2. Uniprot P20248

Application Concentration Tested Antigen Species Retrieval

Western blot 0.1-0.5 μg/ml Human, Rat -

Immunohistochemistry 0.5-1 µg/ml Human By Heat (Parafin-embedded Section) Immunohistochemistry(Frozen 0.5-1μg/ml Human Section) Immunocytochemistry/ 2μg/m Human Immunofluorescence 1-3μg/1x106 Flow Cytometry Human cells, Other applications have not been tested. Optimal dilutions should be determined by end users.

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Preparation and storage

Reconstitution Add 0.2ml of distilled water will yield a concentration of 500ug/ml.

Concentration Add 0.2ml of distilled water will yield a concentration of 500ug/ml.

Storage At -20˚C for one year. After reconstitution, at 4˚C for one month. It can also be aliquoted and stored frozen at - 20˚C for a longer time. Avoid repeated freezing and thawing.

Relevant detection systems Nordic BioSite provides a series of assays reacting with primary antibodies.

Background

Cyclin A2, known as CCNA2, is mapped to 4q27. The protein encoded by this belongs to the highly conserved cyclin family, whose members are characterized by a dramatic periodicity in protein abundance through the cell cycle. Cyclins function as regulators of CDK kinases. Different cyclins exhibit distinct expression and degradation patterns which contribute to the temporal coordination of each mitotic event. In contrast to cyclin A1, which is present only in germ cells, this cyclin is expressed in all tissues tested. This cyclin binds and activates CDC2 or CDK2 kinases, and thus promotes both cell cycle G1/S and G2/M transitions. And Cyclin A2 is synthesized at the onset of S phase and localizes to the nucleus, where the cyclin A2-CDK2 complex is implicated in the initiation and progression of DNA synthesis. Phosphorylation of and MCM4 by the cyclin A2-CDK2 complex prevents re-replication of DNA during the cell cycle

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Images

Fig. 1 IHC analysis of Cyclin A2 using anti-Cyclin A2 antibody (ABB-2326). Cyclin A2 was detected in paraffin-embedded section of Human Lung Cancer Tissue. Heat mediated antigen retrieval was performed in citrate buffer (pH6, epitope retrieval solution) for 20 mins. The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1μg/ml rabbit anti-Cyclin A2 Antibody (ABB-2326) overnight at 4°C. Biotinylated goat anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Streptavidin-Biotin-Complex (SABC)(Catalog # KBB-92FDB1) with DAB as the chromogen.

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Fig.2 IF analysis of Cyclin A2 using anti- Cyclin A2 antibody (ABB-2326)) Cyclin A2 was detected in immunocytochemical section of U20S cell. Enzyme antigen retrieval was performed using IHC enzyme antigen retrieval reagent (RBB-110) for 15 mins. The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2μg/mL rabbit anti- Cyclin A2 Antibody (ABB-2326) overnight at 4°C. Cy3 Conjugated Goat AntiRabbit IgG (ABB-721) was used as secondary antibody at 1:100 dilution and incubated for 30 minutes at 37°C. Visualize using a fluorescence microscope and filter sets appropriate for the label used.

Fig.3 Western blot analysis of Cyclin A2 using anti-Cyclin A2 antibody (ABB-2326). Electrophoresis was performed on a 5-20% SDS-PAGE gel at

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70V (Stacking gel) / 90V (Resolving gel) for 2-3 hours. The sample well of each lane was loaded with 50ug of sample under reducing conditions. Lane 1: Rat Skeletal Muscle Tissue Lysate Lane 2: HELA Whole Cell Lysate Lane 3: COLO320 Whole Cell Lysate Lane 4: HEPG2 Whole Cell Lysate Lane 5: MCF-7 Whole Cell Lysate After Electrophoresis, proteins were transferred to a Nitrocellulose membrane at 150mA for 50-90 minutes. Blocked the membrane with 5% Non-fat Milk/ TBS for 1.5 hour at RT. The membrane was incubated with rabbit anti-Cyclin A2 antigen affinity purified polyclonal antibody (Catalog # ABB-2326) at 0.5 μg/mL overnight at 4°C, then washed with TBS-0.1%Tween 3 times with 5 minutes each and probed with a goat anti-rabbit IgG-HRP secondary antibody at a dilution of 1:10000 for 1.5 hour at RT. The signal is developed using an Enhanced Chemiluminescent detection (ECL) kit (Catalog # KBB-238) with Tanon 5200 system. A specific band was detected for Cyclin A2 at approximately 49KD. The expected band size for Cyclin A2 is at 49KD.